Appl Microbiol Biotechnol (2008) 81:1322 DOI 10.

1007/s00253-008-1590-3

MINI-REVIEW

Synthesis and application of dipeptides; current status and perspectives

Makoto Yagasaki & Shin-ichi Hashimoto

Received: 18 April 2008 / Revised: 22 June 2008 / Accepted: 23 June 2008 / Published online: 16 September 2008 # Springer-Verlag 2008

Abstract The functions and applications of L--dipeptides (dipeptides) have been poorly studied compared with proteins or amino acids. Only a few dipeptides, such as aspartame (L-aspartyl-L-phenylalanine methyl ester) and Lalanyl-L-glutamine (Ala-Gln), are commercially used. This can be attributed to the lack of an efficient process for dipeptide production though various chemical or chemoenzymatic method have been reported. Recently, however, novel methods have arisen for dipeptide synthesis including a nonribosomal peptide-synthetase-based method and an Lamino acid -ligase-based method, both of which enable dipeptides to be produced through fermentative processes. Since it has been revealed that some dipeptides have unique physiological functions, the progress in production methods will undoubtedly accelerate the applications of dipeptides in many fields. In this review, the functions and applications of dipeptides, mainly in commercial use, and methods for dipeptide production including already proven processes as well as newly developed ones are summarized. As aspartame and Ala-Gln are produced using different industrial processes, the manufacturing processes of these two dipeptides are compared to clarify the characteristics of each procedure. Keywords Dipeptide . L-Amino acid -ligase . NRPS . Aspartame . L-Alanyl-L-glutamine
M. Yagasaki (*) : S.-i. Hashimoto Technical Research Laboratories of Kyowa Hakko Kogyo Co., Ltd., 1-1 Kyowa-cho, Hofu 747-8522, Japan e-mail: m.yagasaki@kyowa.co.jp S.-i. Hashimoto e-mail: shashimoto@kyowa.co.jp

Introduction There have been numerous studies on the function, application, and preparation of proteins and their components, amino acids. In contrast, L--dipeptides (dipeptides), the simplest peptide bond product of two amino acids, have been poorly investigated. One of the major reasons is due to the low availability of dipeptides because of the lack of cost-effective manufacturing processes. However, information on unique and interesting functions of dipeptides has still been accumulating. Establishing an efficient process for dipeptide production is expected to boost the exploration and development of the value of dipeptides. In this article, the functions and applications of dipeptides are summarized and current and newly developed technologies for dipeptide production are reviewed. Though there are dipeptides which contain unproteinogenic amino acids or are cyclic structures (diketopiperazine) in nature (Hashimoto 2006), we will focus on linear dipeptides of proteinogenic amino acids and their simple derivatives in this review.

Function and application of dipeptides The function of dipeptides can be considered from two viewpoints, as a derivative of amino acid(s) and as the dipeptide itself. The former viewpoint is easy to understand because dipeptides and the constitutive amino acids have different physicochemical properties but should share the same physiological effects since dipeptides are degraded into the individual amino acids in organisms. For example, L-glutamine (Gln) is heat labile while the dipeptide L-alanyl-L-glutamine (Ala-Gln) is much more tolerant to high temperature (Stehle et al. 1984; Roth et al. 1988). Solubility is another obvious example. Tyr is practically insoluble but Ala-Tyr can be

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Appl Microbiol Biotechnol (2008) 81:1322

dissolved up to 14 g/L (Furst 2001). It is interesting to point out that some dipeptides are much more soluble than each of the constitutive amino acids; the solubilities of Ala and Gln are 89 and 36 g/L, respectively, whereas that of Ala-Gln is 586 g/L (Furst 2001). Based on these properties and the fact that Ala-Gln and Gly-Tyr are rapidly degraded into the individual amino acids once taken into the human body (Albers et al. 1988; Abumard et al. 1989; Frust et al. 1997), they are used as components of patient infusions. Some dipeptides have unique functions which cannot be found in the constitutive amino acids. Dipeptides which have commercial applications based on their unique functions are listed in Table 1. Carnosine (-alanyl-His) and the related dipeptide anserine (-alanyl-N-methyl-His) have been found to exist in a wide range of tissues of mammalian, bird, or fish origin (Gulewitsch and Amiradzibi 1900; Hines and Sutfin 1956). Many functions have been anticipated to these dipeptides, such as antioxidation (Guiotto et al. 2005) and maintenance of cellular pH (Begum et al. 2005). Reflecting these possible functions, the dipeptides and their derivatives have been used in several ways. They are employed in sport nutrition based on the fact that the muscle of a fastswimming fish, the skipjack tuna, contains these dipeptides in relatively high concentrations (Suzuki et al. 1987). Zinc carnosine and N-acetyl carnosine are used as an antiulcer drug (Cho et al. 1991) and as an agent for cataracts (Babizhayev et al. 2001), respectively. Research into the taste of dipeptides also has a long history. The taste of synthetic dipeptides were examined and most of them were reported to be bitter (Schiffman 1976; de Armas et al. 2004). The relationship between bitterness and the physicochemical properties of the dipeptides has captured researchers interest. From the view point of commercial applications, aspartame (Asp-Phe methyl ester) is the only one of outstanding importance. More than 19,000 metric tons of aspartame, which is 180 times as sweet as sugar (Cloninger and Baldwin 1970; Ager et al. 1998), is used annually around the world as a low-calorie sweetener.

Recently, the antihypertensive effect of dipeptides has attracted researchers attention (Kitts and Weiler 2003). Some extracts or hydrolysates of fish meat, seaweed, or mushrooms have been reported to exert a blood-pressurelowering effect and the active agents were identified as several kinds of dipeptides, such as Ile-Tyr, Lys-Trp, Val-Tyr, and Ile-Trp. The antihypertensive effects of these dipeptides have been demonstrated to be derived from their inhibitory effect on angiotensin-I-converting enzyme (Kitts and Weiler 2003; Matsufuji et al. 1994; Sato et al. 2002; Yokoyama et al. 1992). The extracts or hydrolysates containing these dipeptides have been approved as foods for specified health uses in Japan. Apart from these industrially applied dipeptides, there are several dipeptides not used practically but whose functions are known. Kyotorphin (Arg-Tyr) was isolated from bovine brain and shown to have analgesic effects (Takagi et al. 1979). A synthetic dipeptide, Lys-Glu, was reported to have antitumor activity (Khavinson and Anisimov 2000). Leu-Ile was described to have a neuroprotective effect (Nitta et al. 2004). Tyr-Gly was shown to enhance proliferation of peripheral blood lymphocytes (Kayser and Meisel 1996). It should be also be mentioned that transport mechanisms for dipeptides and amino acids in human intestine are different (Adibi 1997). This implies that a dipeptide and the corresponding amino acids may exert different nutritional impacts on the human body when taken orally.

Technologies for dipeptide synthesis Various ways are known for producing a dipeptide or to form a peptide bond. They are categorized into three methods: chemical synthesis, chemoenzymatic synthesis, and enzymatic synthesis (in this review, chemoenzymatic synthesis is defined as the method which uses an enzyme and at least one protected amino acid as the substrate).

Table 1 Commercially applied dipeptides and their unique functions Compound Aspartame Ala-Gln Gly-Tyr Carnosine N-Acetyl carnosine Val-Tyr Usage Sweetener Patient infusion Patient infusion Sport nutrition Antiulcer (zinc salt) Prevention of cataracts Health food (antihypertensive) Commercial form Pure Pure Pure Crude Pure Crude extract Reference Ager et al. 1998 Frust et al. 1997 Albers et al. 1988 Begum et al. 2005 Cho et al. 1991 Babizhayev et al. 2001 Sato et al. 2002

Appl Microbiol Biotechnol (2008) 81:1322

15

Chemical synthesis While myriad methods are known (for review, Katsoyannis and Ginos 1969; Nilsson et al. 2005), the principal scheme of chemical synthesis of dipeptides is as follows (for example, see Tables 3 and 4): 1. All of the functional groups except for those involved in making the peptide bond of the amino acids are protected. 2. The free carboxy group of the protected amino acid is activated. 3. The activated amino acid is reacted with the other protected amino acid. 4. All the protecting groups on the dipeptide are removed. The advantages of chemical synthesis are summarized as follows: (1) all kinds of dipeptides can be synthesized by choosing appropriate protecting groups and activating reagents; (2) the yield is usually high; (3) the procedure is easy to carry out in a small scale. On the other hand, the following disadvantages of chemical synthesis can be pointed out. (1) The cost of synthesis is relatively expensive because of the necessity of employing many reaction steps and reagents. (2) There is a risk of racemization during reactions. (3) A harmful reagent is sometimes needed. Because of the balance of these advantages and disadvantages, chemical synthesis has been used mainly to fulfill the demands of research laboratories. Chemoenzymatic synthesis Peptide-bond-hydrolyzing enzymes, such as proteases and esterases, can be used to catalyze the reverse reaction, i.e., peptide bond formation between connection of two amino acids. This approach extends back to late nineteenth century (Henriques and Gjaldbak 1911). In the 1930s, Bergmann and
Fig. 1 a, b Schematic reactions of chemoenzymatic dipeptide synthesis

Fraenkel-Conrat (1937, 1938) first demonstrated the synthesis of well-defined peptides using proteolytic enzymes. Since then, several hundred reports on this area have been published. Enzymatic synthesis has many advantages over chemical one, including strict stereoselectivity and more mild conditions. To direct the order of the connection of the amino acids and to drive the synthetic reaction, protection of the substrate amino acids (at least one substrate) is required. Two types of processes with different reaction mechanisms are known, an equilibrium-controlled (thermodynamically controlled) process or a kinetically controlled process (Bordusa 2002; Sinisterra and Alcantara 1993; Kumar and Bhalla 2005). The equilibrium-controlled process is based on the reverse reaction of a protease or an esterase (Fig. 1a). As expected from the nature of the enzymes, the equilibrium of the reaction is on the side of the hydrolysis products under physiological conditions. To drive the equilibrium towards peptide synthesis, some intervention is necessary. When the solubility of the product is much less than those of the substrates, precipitation can be used. Precipitation of the dipeptide product removes the product from the reaction equilibrium, promoting the synthetic direction. Other than precipitation, several techniques, such as conducting the reaction under a large excess of the substrate(s) or under biphasic conditions in which the transfer of the product from aqueous catalyst phase to immiscible phase promotes synthetic reaction, have been reported (for review, Lombard et al. 2005). The kinetically controlled process depends on the fact that a mildly activated C-terminal ester (or amide) rapidly acylates a serine or cysteine protease. The acyl enzyme intermediate undergoes a rate-limiting competitive deacylation by water and by an added nucleophile (the other amino acid) to give a transient accumulation of the product dipeptide (Fig. 1b). Since the protease slowly hydrolyzes

(a) Equilibrium-controlled process

O R1 P1 NH OH + R2
NH2 O OH

O R1 P1 NH NH R2

O OH

(b) Kinetically controlled process

O R1 NH2 OX O

H2 O
Enz

O R1 NH2
O

+ Enz-H

R1 NH2

+ XOH

OH + XOH

Enz-H

O H2N R2 O R1 NH2 Enz H2 N R2 O OH OH

H2 O

H2N R2

OH O R1 O NH NH2 R2 OH

Enz-H

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Appl Microbiol Biotechnol (2008) 81:1322

the dipeptide formed, the accumulation of the dipeptide is temporary. Because of this reaction mechanism, the product yield depends on the velocities of the attack by water and the nucleophile to the acyl enzyme and of degradation of the dipeptide by the protease itself. Thus, as well as in properties of the enzyme itself, the reaction conditions, such as pH, ionic strength, and concentration of the nucleophile, are crucially important. For more details, please refer to the excellent reviews (Bongers and Heimer 1994; Morihara 1987; Schellenberger and Jakubke 1991). Enzymatic synthesis While the ribosome system is the ubiquitous equipment for peptide formation in organisms, other enzymatic machineries which conduct peptide syntheses have been found in nature. These include nonribosomal peptide synthetase (NRPS, see below), polyglutamine synthase (Ashiuchi and Misono 2002), cyanophycin synthetase (Aboulmagd et al. 2001), glutathione synthase (Meister 1974), D-alanine-Dalanine ligase (Ddl, Walsh 1989), and L-amino acid ligase (Lal, see below). Activities other than the ribosomal system are specific for their own products. From the view point of the way to activate the substrate amino acid(s), these activities can be divided into two groups. One way is via aminoacyl-adenosine monophosphate (AMP) and the other way is via aminoacyl phosphate. The ribosomal system and NRPS belong to the former group whereas cyanophycin synthetase, glutathione synthase, Ddl, and Lal belong to the latter group. Both ways have been proposed for polyglutamine synthase (Ashiuchi and Misono 2002; Candela and Fouet 2006). Since these naturally occurring peptide-synthesizing activities use unprotected amino acids as their substrates and catalyze only peptide-forming reactions, they seem to be ideal catalysts for dipeptide synthesis. The first such attempt was conducted in 1980. Doel et al. (1980) expressed synthetic genes coding for a protein consisted of about 150 repeats of Asp-Phe in Escherichia coli. Unfortunately, this strategy was not practical because of the low productivity and simultaneous appearance of Phe-Asp along with Asp-Phe when the produced polymer was cleaved by proteases. In the 1990s, progress in a NRPS study provided researchers with another approach. And recently, Lal has been demonstrated to be useful for dipeptide production. These emerging approaches and other possibilities are reviewed below. NRPS process NRPSs have been found in various microorganisms such as bacteria and fungi. They are responsible for the syntheses of a wide array of therapeutically important peptides such as vancomycin, gramicidin S, and cyclosporine. The

enzymes are huge multifunctional proteins and are made up of a series of modules, each of which takes charge of adding one amino acid to a growing peptide. Each module contains at least three enzymatic units called domains (Fig. 2). An adenylation domain (A-domain) recognizes the substrate amino acid and activates it as an aminoacyl-AMP accompanied with the hydrolysis of adenosine triphosphate (ATP) to AMP and pyrophosphate. Subsequently, the activated amino acid is transferred to 4-phophopantetheine moiety of the thiolation domain (T-domain) with the release of AMP. Then the adjacent condensation domain (Cdomain) catalyzes the formation of the peptide bond. Finally, the thioesterase domain (Te-domain) catalyzes the release of the product peptide from the enzyme protein. For more details on NRPS, please refer to the comprehensive reviews (Finking and Marahiel 2004; Sieber and Marahiel 2005). Modular manipulation of NRPS has been applied to dipeptide synthesis. Doeckel and Marahiel designed artificial NRPSs by combining the A-domain, which recognizes Ile, from the bacitracin-biosynthetic NRPS in Bacillus licheniformis and the A-domain, which recognizes Leu, from the tyrocidine-biosynthetic NRPS in Bacillus brevis (Doekel and Marahiel 2000). The artificial dimodular NRPS was expressed in E. coli simultaneously with 4phosphopantetheinyl transferase, which is necessary to make NRPS holoenzyme. The purified enzyme was demonstrated to produce Ile-Leu in the presence of Ile, Leu, and ATP. A similar strategy was applied to create NRPSs for Phe-Leu, Ile-Phe (Doekel and Marahiel 2000),

O R1 NH2 OH

+ ATP

R1 NH2

O AMP

+ PPi

O S R1 NH2

AMP

T-domain C-domain O
S

R2 NH2

Te-domain

O S HN R2

O R1 NH2

O HO HN R2

O R1 NH2

Fig. 2 NRPS-catalyzed peptide bond formation

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17

Phe-Ala (Dieckmann et al. 2001), D-Phe-Pro (Keller and Schauwecker 2003), and Asp-Phe (see the latter section). From bioinformatics data and biochemical or structural data, the selectivity-conferring nonribosomal code of the A-domain has been determined (Stachelhaus et al. 1999; Rausch et al. 2005), enabling researchers to design an artificial enzyme for a desired peptide. But, practically, there are still problems to overcome. One problem is Cdomain selectivity. While substrate specificity of NRPS is basically determined by the A-domains, the C-domains also have selectivity, which are important for the control of the directionality of the peptide synthesis (Belshaw et al. 1999; Linne and Marahiel 2004). Therefore, a suitable combination of A- and C-domains needs to be chosen. The other problem is the low activities of the artificially created enzymes. This may be the reason for the fact that there have been a small number of reports on peptide production by the living cells expressing engineered NRPS (Stachelhaus et al. 1995; de Ferra et al. 1997; Symmank et al. 2002; Mootz et al. 2002). Product yields in most of the living-cell studies also remained quite low. How to fuse the domains has been found to be important to optimize the interaction of domains (Linne and Marahiel 2004). Thus, in contrast with the succinctness of its principle, NRPS engineering needs a lot of know-how to be applied practically. Lal process Lal was discovered by an in silico screening for a new activity to catalyze a dipeptide formation (Tabata et al. 2005). The only gene found, ywfE in Bacillus subtilis, was expressed in E. coli and confirmed to have the expected activity, ligating Ala and Gln in an ATP-dependent manner. From the amino acid sequence and biochemical data, the enzyme was determined to belong to the ATP-dependent carboxylate-aminethiol ligase superfamily, which uses acyl phosphate as the reaction intermediate (Galperin and Koonin 1997; Fig. 3). The following characteristics of the
O R1 NH2 OH O

+ ATP

R1 NH2

Pi

+ ADP

Lal

H2N R2 O

OH

R1 NH2
Fig. 3 Lal-catalyzed dipeptide formation

O NH R2 OH

Pi

enzyme were reported. (1) It forms only dipeptides. Tripeptides or longer peptides were never detected as the reaction product. (2) It can take various kinds of amino acids as the substrates but has certain selectivity. Acidic or basic amino acids do not react. The order of the amino acids is also directed, for example, Ala-Gln can be formed but Gln-Ala cannot. Consequently, 44 kinds of dipeptides were confirmed to be synthesized. (3) The enzyme dose not accept D-amino acids. Two types of processes for dipeptide production utilizing Lal have been described, the resting cell reaction process and the direct fermentation process. The resting cell reaction process is a coupling reaction of Lal and an ATP regeneration reaction. Detergent-treated E. coli cells expressing Lal from B. subtilis and polyphosphate kinase from Rhodobacter sphaeroides was reported to produce several kinds of dipeptides (Ala-Met, Ala-Val, Ala-Ile, Ala-Leu, Gly-Met, and Gly-Phe) by incubating the corresponding amino acids and polyphosphate (Ikeda et al. 2006). Ala-Met gave the highest titer, 127.9 mM (28 g/L), from 200 mM each of Ala and Met. Any dipeptides within the product spectrum of Lal can be produced by changing the substrate amino acids. One can imagine that a Lal-expressing organism would produce some dipeptides because Lal takes unprotected amino acids. This is the conceptual idea of the direct fermentation. But simply expressing Lal in E. coli resulted in no accumulation of dipeptides (Tabata and Hashimoto 2007). This is attributed to two problems, first, the relatively low affinity of Lal for amino acids and, second, the dipeptide-degrading activity of the host cells. To overcome these problems, some metabolic engineering, such as enhancing the metabolic flux to the substrate amino acids and reducing the degradation activity, are necessary. Ala-Gln fermentation is a successful example (see the latter section). Some other producer strains for Ala-Met and Thr-Phe, respectively, were also reported (Tabata and Hashimoto 2005). Obviously, the direct fermentation method is the most cost-effective for dipeptide manufacturing since it does not need even the substrate amino acids. YwfE seemed to be an orphan enzyme since there has been no clear homolog of YwfE in the public database except for BacD from Bacillus amyloliquefaciens, which encodes a protein that is 97% identical with YwfE. Recently, however, several homologs have been found. Proteins encoded by rsp1486 in Ralstonia solanacearum and bl00235 in B. licheniformis were reported to have only 29% and 28% identity with YwfE, respectively, but have Lal activities (Kino et al. 2006, 2007). A gene involved in the biosynthesis of rhizocticin A, a peptidic antibiotic produced by B. subtilis, was shown to possess Lal activity (Kino et al. 2008). Interestingly, these newly found YwfE

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Appl Microbiol Biotechnol (2008) 81:1322

homologs were shown to have different product spectrums from that of YwfE from B. subtilis (Table 2).

Approaches for specific targets Aspartame and Ala-Gln are the rare cases of commercialized dipeptides. Several manufacturing methods have been proposed for each dipeptide. Comparing those will help to understand the pros and cons of each process. They will also show that the choice of an industrial manufacturing process depends not only on the efficiency of the dipeptide-forming reaction but also on the controllability of by-product(s) and economics of the total process. Aspartame Two processes have been commercially used, chemical synthesis and chemoenzymatic synthesis (Table 3) while there have been hundreds of patents and reports on improvements or modifications of these principal processes. The chemical synthesis of aspartame is basically carried out as follows. (1) Phe is reacted with methanol to yield Phe methyl ester (PheOM). (2) Asp is modified to an N-protected aspartic anhydride such as N-carbobenzoxyaspartic anhydride or N-formyl-aspartic anhydride. Industrially, the cheaper formyl compound would be preferable. (3) PheOM and N-protected aspartic anhydride are reacted to form N-protected Asp-PheOM. (4) N-protected AspPheOM is treated with acid to get Asp-PheOM, aspartame. The overall yield mainly depends on the yield of the

condensation step 3, which has been reported to be 65~98% (Ariyoshi et al. 1974a, b; Albini et al. 1985). The biggest problem of the chemical synthesis is the by-production of -Asp-PheOM, which exhibits a bitter taste (Albini et al. 1985). Much effort have been paid to reduce the formation of this by-product (Albini et al. 1985; Yukawa et al. 1994; Hill et al. 1991). The chemoenzymatic process employs thermolysin from Bacillus thermoproteolyticus (Lombard et al. 2005; Isowa et al. 1979). The synthetic route is very similar to the chemical synthesis. But, thanks to the selectivity of the enzyme, the process is free from the -form and can use DL-Phe instead of L-Phe as the starting material. N(benzoyloxycarbonyl)-Asp (Z-Asp) and DL-PheOM are prepared and connected through the action of thermolysin. In spite of the equilibrium-controlled mechanism, Z-AspPheOM synthesis by the enzyme proceeds efficiently because the product Z-Asp-PheOMe forms insoluble salt with the remaining D-PheOM (Oyama et al. 1987). A very high yield (95%) in the condensation reaction has been described (Nakanishi et al. 1985, 1990). After separating ZAsp-PheOM and D-PheOM, the latter compound is racemized to DL-PheOM and reused. While the overall process is sophisticated, the decrease in the enzymatic activity is a drawback of the process. To improve it, many attempts have been investigated such as immobilization (Oyama et al. 1987; Nakanishi et al. 1985, 1990), enzyme engineering (Inouye et al. 2007), or use of a molecular imprinted polymer (Ye et al. 1999). There have also been reports on the enzymatic synthesis of aspartame from unprotected Asp and PheOM (Table 3), but the yields remained low (Francois et al. 1990).

Table 2 Product spectrums of YwfE and its homologs C-terminus Gly N-terminus Gly Ala Ser Cys Thr Leu Met Phe Gln His Arg Y Y Y Y B B R, B R R R Z B R R Z Ala Ser Cys Y Y Y, R Y, R Thr Val Leu Y Y Y Y Y Ile Met Y Y Y Y Y Y, R R R R Phe Y Y Y Y Tyr Y Y Y Trp Gln Y Y Y Y Y R Y Y Asn His Arg

Y, R Y, R

Y Y, R Y

Y Y Y

Y Y

Y Y Y

Y Y Y

Y Y

Y Y Y

R Z

Amino acids able to be accepted at the N-terminus are listed in the file. The ones able to be accepted at the C-terminus are listed in the line. Each product were confirmed by HPLC or NMR analysis. Y YwfE of B. subtilis, Z a gene product involved in rhizocticin biosynthesis, B protein encoded by bl00235 in B. licheniformis, R; protein encoded by rsp1486 in R. solanacearum.

Appl Microbiol Biotechnol (2008) 81:1322 Table 3 Methods for aspartame production
Category (a) Chemical
Asp
H2N
O O N H O O

19

Schematic scheme

Advantage / disadvantage* High yield

CO2H H N O

Reference Ariyoshi et al. 1974a,b, Albini et al. 1985

O N H

CO2H H N O

CO2CH3

H2N
O O N H N H CO2H CO2CH3

CO2CH3

CO2CH3

Phe

By-product formation of -form High yield, high sterospecifcity Lombard et al. 2005, Isowa et al.
CO2CH3

-form
(b) Chemoenzymatic
O O N H CO2H CO2H
O O N H CO 2H N O

Asp

+H3N
CO2CH3

CO 2CH3
O O N H

CO 2H H N O

1979,

Nakanishi et al. Decrease in enzyme activity 1990

H2N

CO2 CH 3 CO2 H H N O

DL-Phe
H2 N

CO2CH 3

(c) Chemoenzymatic

Asp
H2N CO2 CH 3 H2 N

CO2H H N O

CO 2CH3

High Francois et al. stereospecificity, 1990 simple process

Phe

Low yield (d) Enzymatic (+chemical)

a

Cheap raw
Asp + Phe
Asp-Phe Asp-PheOM

Bachman et al. 1976, Duerfahrt et al. 2003

materials in vitro level

Upper section, advantage; lower section, disadvantage

Aspartame can be made in a different way, through the selective esterification of Asp-Phe (Bachman et al. 1976). As an attempt to this end, the enzymatic synthesis of AspPhe has been reported. Duerfahrt and coworkers created artificial NRPSs containing the A-domain for Asp from surfactin synthetase and the A-domain for Phe from tyrocidine synthetase (Duerfahrt et al. 2003). Six artificial genes with different fusion points and/or Te domains were constructed. These NRPSs were purified and confirmed to have the desired activity (Table 3). While all of them were capable of synthesizing Asp-Phe, significant differences in the activity depending on the fusion strategy were observed, indicating the importance of the design of the artificial NRPS. Ala-Gln Since the effectiveness of Ala-Gln as a component of patient infusions has been established (Furst et al. 1997; Goeters et al. 2002), the dipeptide has been used in medical fields. The following four processes (Table 4) or their modified versions are used commercially. While standard chemical synthetic methods can yield the dipeptide, a chemical liquid-phase peptide synthesis via N-

carboxyanhydride intermediate (Furst et al. 1985) is used for Ala-Gln production (Table 4). Ala is reacted with carbonyl chloride to form N-carboxyalanine anhydride followed by condensation with Gln. The Ala-Gln carbamate formed is treated by an acid to yield Ala-Gln. As Ncarboxyalanine anhydride is highly reactive, the condensation rate is high. On the other hand, by-products such as Ala-Ala-Gln and D-Ala-Gln are also formed. The other problem of this method is the use of carbonyl chloride, which is the famous poison gas, phosgene. Sano et al. (2000) designed an alternative chemical route. D-2-chloropropionic acid was subjected to the Schottenn-Baumann reaction with Gln to yield D-2chloropropionyl-Gln. Ala-Gln was obtained by the ammonolysis reaction of the product (Table 4). Some by-products including Ala-Glu were detected, but they could be removed by recrystallization. A chemoenzymatic process has also recently been developed (Table 4). Yokozeki and Hara screened for the activity to synthesize Ala-Gln from Ala methyl ester (AlaOM) and Gln and found an enzyme from Empedobacter brevis. The purified enzyme was demonstrated to produce 83 mM of Ala-Gln from 100 mM of AlaOM and 200 mM of Gln (Yokozeki and Hara 2005). In the absence of Gln, the

20 Table 4 Methods for Ala-Gln production

Category (a) Chemical
COCl2
H2N CO 2H
HM O O O O NH 2 H N CO2 H CONH 2

Appl Microbiol Biotechnol (2008) 81:1322

Schematic scheme

Advantage / disadvantage* High yield Ala-Ala-Gln formation, Use of phosgen High

Reference Frust et al. 1985

Gln

(b) Chemical
Cl CO 2H

Sano et al. 2000

SOCl2 + Gln

Cl

H N O CO 2H

CONH 2

NH3

NH 2 O

H N CO 2 H

CONH 2

stereospecificty Ala-Glu formation Simple & easy Yokozeki and Hara 2005

(c) Chemoenzymatic
H 2N CO 2H H 2N O

NH 2 O

H N CO 2H

CONH 2

process Low yield, Ala-Ala-Gln formation

Gln

(d) Enzymatic (fermentation)

Glucose + NH3
NH 2 O H N CO2 H CONH 2

Cheap raw material, easy process Ala-Ala formation

Tabata and Hashimoto 2005

Upper section, advantage; lower section, disadvantage

enzyme hydrolyzed Ala-Gln, suggesting a kinetically controlled mechanism. Recently, a Lal-based enzymatic process has been established (Tabata and Hashimoto 2007). Tabata and Hashimoto constructed a recombinant E. coli strain producing Ala-Gln without supplying Ala and Gln. To enhance metabolic fluxes to the substrate amino acids, Gln biosynthesis was deregulated by destroying glnE and glnB genes and alanine dehydrogenase (Ald) from B. subtilis was coexpressed with Lal. To reduce dipeptide degradation, genes for several dipeptidases (PepA, PepB, pepD, and PepN) and the dipeptide import system (Dpp) were disrupted in combination. Lal and Ald were expressed in the host strain under a stationary-phase specific promoter since the synthesis of Lal hampered cell growth. Fed batch cultivation of the recombinant strain in a 5-L jar fermentor on a glucose-ammonium medium resulted in the accumulation of Ala-Gln (100 mM) in the cultivation supernatant (Table 4). No tripeptides or D-amino acid containing dipeptides were detected. Ala-Ala was also produced, but it can be separated by chromatography or crystallization. Perspective It is interesting that several production methods have been employed for commercial production of aspartame or Ala-

Gln, illustrating researchers originality and ingenuity. Each process has its own advantages and disadvantages (Table 3 and 4). These facts indicate that dipeptide manufacturing is still in the early stages of the technology evolution, in which processes converges to the most competitive methodology. Fermentative production of dipeptides based on NRPS or Lal could become such the ultimate method because it is likely the most cost-efficient and environmentally friendly. Since dipeptide fermentation has just emerged, it needs to be more thoroughly studied to be applied widely; including metabolic flow control for the substrate amino acids and suppression of undesired byproducts. In addition, which enzyme of NRPS and Lal should be used must be decided. The possible product spectrum of the NRPS system is much wider than the Lal system whereas the Lal system is easier to be practically applied. Finding of Lal homologs suggests feature expansion of the product by the Lal-based fermentation. Development of a new function or application and development of an efficient production method are in a mutually promoting relationship. The recent increase in the interest in the functions of dipeptides and the appearance of new processes for production imply that the exploration of the dipeptide world both in applications and manufacturing technology will be much accelerated over the next decade.

Appl Microbiol Biotechnol (2008) 81:1322

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